Cell adhesion molecules regulate contractile ring-indepen- dent cytokinesis in Dictyostelium discoideum

To investigate the roles of substrate adhesion in cytokinesis, we established cell lines lacking paxillin （PAXB） or vineulin （VINA）, and those expressing the respective GFP fusion proteins in Dictyostelium discoideum. As in mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like complexes on the cell bottom, paxB^- cells in suspension grew normally, but on substrates, often failed to divide after regression of the furrow. The efficient cytokinesis of paxB^- cells in suspension is not because of shear forces to assist abscission, as they divided normally in static suspension culture as well. Double knockout strains lacking mhcA, which codes for myosin II, and paxB or vinA displayed more severe cytokinetic defects than each single knockout strain. In mitotic wild-type cells, GFP-PAXB was diffusely distributed on the basal membrane, hut was strikingly condensed along the polar edges in mitotic mhcA^- cells. These results are consistent with our idea that Dictyostelium displays two forms of cytokinesis, one that is contractile ringdependent and adhesion-independent, and the other that is contractile ring-independent and adhesion-dependent, and that the latter requires PAXB and VINA. Furthermore, thatpaxB- cells fail to divide normally in the presence of substrate adhesion suggests that this adhesion molecule may play additional signaling roles.

Calmodulin regulates the post-anaphase reposition of centrioles during cytokinesis

A transient postanaphase repositioning of the centriole is found to control the completion of cytokinesis. Using agreen fluorescent protein-calmodulin fusion protein as a living cell probe, we have previously found that calmodulin isassociated with the initiation and progression of cytokinesis. In this study, we further studied the effect of calmodulinon the repositioning of the centriole and subsequent cell cycle progression. When activity of calmodulin is inhibited, theregression of the centriole from the intercellular bridge to the cell center is blocked, and thus the completion of celldivision is repressed and two daughter cells are linked by longer cell bridge in perturbed cells. W7 treatment duringcytokinesis also results in unfinished cytokinesis and stopped G1 phase. These results suggest that calmodulin activity isrequired for centriole repositioning and can affect the completion of cytokinesis and cell cycle progression.

Effects of polar cortical cytoskeleton and unbalanced cortical surface tension on intercellular bridge thinning during cytokinesis

To probe the contributions of polar cortical cytoskeleton and the surface tension of daughter cells to intercellular bridge thinning dynamics during cytokinesis,we applied cytochalasin D（CD） or colchicine（COLC） in a highly localized manner to polar regions of dividing normal rat kidney（NRK） cells.We observed cellular morphological changes and analyzed the intercellular bridge thinning trajectories of dividing cells with different polar cortical characteristics.Global blebbistatin（BS） application was used to obtain cells losing active contractile force groups.Our results show that locally released CD or colchicine at the polar region caused inhibition of cytokinesis before ingression.Similar treatment at phases after ingression allowed completion of cytokinesis but dramatically influenced the trajectories of intercellular bridge thinning.Disturbing single polar cortical actin induced transformation of the intercellular bridge thinning process,and polar cortical tension controlled deformation time of intercellular bridges.Our study provides a feasible framework to induce and analyze the effects of local changes in mechanical properties of cellular components on single cellular cytokinesis.

Eczema herpeticum vs dermatitis herpetiformis as a clue of dedicator of cytokinesis 8 deficiency diagnosis:A case report

BACKGROUND Dedicator of cytokinesis 8(DOCK 8) deficiency,also known as autosomal recessive hyper immunoglobulin E(IgE) syndrome,is a combined immunodeficiency disease that was first recognized in 2009.It is caused by genetic alterations(mutations or deletions) in the DOCK 8 gene and is characterized by multiple allergies,elevated IgE levels,and susceptibility to viral and bacterial infections.Early diagnosis is critical to optimize the success of stem cell transplantation.CASE SUMMARY This study reports the case of a pediatric patient with DOCK 8 deficiency who had negative genetic testing using multiplex primary immunodeficiency(PID) panel and whole-exome sequencing(WES) with a next-generation sequencing method.He presented with chronic diarrhea and was managed as celiac disease based on previous negative workup for immunodeficiency and duodenal biopsy.He developed a generalized vesicular rash which was thought to be dermatitis herpetiformis associated with celiac disease.However,it turned out to be Eczema herpeticum based on positive herpes simplex virus from blood and lesions.The diagnosis was re-evaluated after the child was found to have multiple viral,bacterial,and parasitic co-infections(herpes simplex virus,cytomegalovirus,Epstein-Barr virus,Salmonella,and cryptosporidiosis).Re-evaluation with target gene testing with copy number variation(CNV) analysis and Multiplex Ligation Probe Amplification(MLPA) showed a large homozygous deletion in the DOCK 8 gene,confirming the diagnosis of DOCK 8 deficiency.CONCLUSION Targeted gene testing with CNV analysis might detect deletions that can be missed by WES for diagnosing patients with PID.

NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells.Midbody,a protein-dense organelle that forms at the intercellular bridge,is indispensable for success-ful cytokinesis.However,the regulatory mechanism of cytokinesis at the midbody still remains elusive.Here,we unveil a critical role for NudC-like protein 2(NudCL2),a co-chaperone of heat shock protein 90(Hsp90),in cytokinesis regulation by stabilizing regulator of chromosome condensation 2(RCC2)at the midbody in mam-malian cells.NudCL2 localizes at the midbody,and its downregulation results in cytokinesis failure,multinu-cleation,and midbody disorganization.Using iTRAQ-based quantitative proteomic analysis,we find that RCC2 levels are decreased in NudCL2 knockout(KO)cells.Moreover,Hsp90 forms a complex with NudCL2 to stabilize RCC2,which is essential for cytokinesis.RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells.Importantly,ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion,but not vice versa.Together,our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis

The phragmoplast,a structure crucial for the completion of cytokinesis in plant cells,is composed of antiparallel microtubules(MTs)and actin filaments(AFs).However,how the parallel structure of phragmoplast MTs and AFs is maintained,especially during centrifugal phragmoplast expansion,remains elusive.Here,we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein(AtMAC).When AtMAC was deleted,the phragmoplast showed disintegrity during centrifugal expansion,and the resulting phragmoplast fragmentation led to incomplete cell plates.Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis.Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro,and the truncated AtMAC protein,N-CC1,was the key domain controlling the ability of AtMAC.Further analysis showed that N-CC1(51–154)is the key domain for binding MTs,and N-CC1(51–125)for binding AFs.In conclusion,AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion,which is required for complete cytokinesis.

Regulation of Cytokinesis by Exocyst Subunit SEC6 and KEULE in Arabidopsis thaliana

Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.

Rab-Alc GTPase Defines a Population of the Trans-Golgi Network that Is Sensitive to Endosidinl during Cytokinesis in Arabidopsis

In plant cells, Rab-A proteins have been implicated to play important roles in membrane trafficking from the trans-Golgi network （TGN） to the plasma membrane/cell wall and to the newly formed cell plate in cytokinesis. But how different Rab-A proteins may work in the TGN is not well studied. We show here that RAB-Alc defines a population of TGN that is partially overlapped with the VHA-al marked-TGN. Interestingly, the morphology of RAB-Alc defined- TGN is sensitive to endosidin 1 （ES1）, but not to wortmannin. In mitotic cells, RAB-Alc is relocated to the cell plate. We revealed that this process could be interrupted by ES1, but not by wortmannin. In addition, root growth and cytokinesis in root mitotic cells of rab-ala/b/c triple mutant seedlings are hypersensitive to lower concentrations of ES1. ES1 is known to selectively block the transport of several plasma membrane auxin transporters, including PIN2 and AUX1 at the TGN. Together with the known facts that members of Rab-A1 proteins are involved in auxin-mediated responses in root growth and that mutations in TRAPPII, a protein complex that acts upstream of RAB-Alc, also selectively impair the transport of PIN2 and AUX1 at the TGN, we propose that the Rab-Al-mediated trafficking pathways around the TGN, but not Rab-Als directly, are the target of ES1.

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M （mitosis） phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytoldnesis can be morphologically divided into four steps： cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are im- portant players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 （Plkl） is an important kinase that can target many substrates and carry out different functions during mitosis, including cytokinesis. Recent studies are beginning to unveil a closer tie be- tween Plkl and RhoA networks. More specifically, Plkl phosphorylates the centralspindlin complex Cyk4 and MKLPI/CHO1, thus re- cruiting RhoA guanine nucleotide-exchange factor （GEF） Ect2 through its phosphopeptide-binding BRCT domains. Ect2 itself can be phosphorylated by Plkl in vitro. Plkl can also phosphorylate another GEF MyoGEF to regulate RhoA activity. Once activated, RhoA-GTP will activate downstream effectors, including ROCK1 and ROCK2. ROCK2 is among the proteins that associate with Plkl Polo-binding domain （PBD） in a large proteomic screen, and Plkl can phosphorylate ROCK2 in vitro. We review current understandings of the interplay between Plkl, RhoA proteins and other proteins （e.g., NudC, MKLP2, PRC 1, CEP55） involved in cytokinesis, with particular emphasis of its clinical implications in cancer.

The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis

Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between membrane trafficking and cytoskeletal dynamics. In plants, the onset of cytokinesis is characterized by the assembly of a bipolar microtubule array, the phragmoplast, and of a transient membrane compartment, the cell plate. Little is known about the coordination between membrane deposition at the cell plate and the dynamics of phragmoplast microtubules. In this study, we monitor the localization dynamics of microtubule and membrane markers throughout cytokinesis. Our spatiotemporal resolution is consistent with the general view that microtubule dynamics drive membrane movements. Nonetheless, we provide evidence for active sorting at the cell plate and show that this is, at least in part, mediated by the TRAPPII tethering complex. We also characterize phragmoplast microtubule organization and cell plate formation in a suite of cytokinesis-defective mutants. Of four mutant lines with defects in phragmoplast microtubule organization, only morl microtubule-associated mutants exhibited aberrant cell plates. Conversely, the mutants with the strongest impairment in phragmoplast microtubule reorganization are keule alleles, which have a primary defect in membrane fusion. Our findings identify the SEC1/Munc18 protein KEULE as a central regulatory node in the coordination of membrane and microtubule dynamics during plant cytokinesis.

The formation of Ca^(2+) gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos,a localized elevation in intracellular Ca^(2+)([Ca^(2+)]i)at the cleavage furrow has been shown to be essential for cytokinesis.However,the underlying mechanisms for generating and maintaining these[Ca^(2+)]_(i) gradients throughout cytokinesis are not fully understood.In the present study,we analyzed the role of inositol 1,4,5-trisphosphate receptors(IP3Rs)and endoplasmic reticulum(ER)distribution in determining the intracellular Ca^(2+) gradients in early zebrafish blastomeres.Application of the injected Ca^(2+) indicator,Indo-1,showed that during the first cell division a standing Ca^(2+) gradient was formed~35 min after fertilization,with the[Ca^(2+)]_(i) spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus.While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region,ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions.Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca^(2+) uptake within the ER was essential for the formation of a standing Ca^(2+) gradient,and the Ca^(2+) gradient could only be well-established under an optimal stoichiometry of Ca^(2+) uptake and release.Indeed,while inhibition of IP3R Ca^(2+) release blocked the generation of the Ca^(2+)gradient at a lower[Ca^(2+)]_(i) level,both Ca^(2+) release stimulation by inositol 1,4,5-trisphosphate(IP3)injection and ER Ca^(2+) pump inhibition by cyclopiazonic acid also eliminated the Ca^(2+) gradients at higher[Ca^(2+)]_(i) levels.Our results suggest a dynamic relationship between ER-mediated Ca^(2+) release and uptake that underlies the maintenance of the perifurrow Ca^(2+) gradient and is essential for cytokinesis of zebrafish embryos.

Targeting cytokinesis bridge proteins to kill high-CIN type tumors

As a common feature of tumors,chromosomal instability(CIN)not only forces carcinomatous evolution,but also loads cancer cells with extra pressure through a robust imbalance of genome patterning that may be used for cancer treatment.Errors in cytokinesis increase CIN,so cytokinesis components are valuable targets for treating cancer.However,due to the short time span and confined space of cytokinesis bridges,profiling cytokinesis fac-tors is challenging.Taking advantage of engineered ascorbate peroxidase(APEX2),we established a cytokinesis bridge-APEX reaction in living cells.A total of 218 cytokinesis bridge proteins were identified with high relia-bility.Knockdown of cytokinesis bridge genes generated micronuclei that activate the cGAS-pathway and cause apoptosis in cancer cells bearing high CIN rather than low CIN.Thus,our study proposes a strategy for killing high-CIN tumors regardless of tumor type,and provides a proteome resource of cytokinetic bridges for future research.

Septin基因家族与人类疾病

Septin基因家族是一类具有GTPase活性的保守基因家族,参与细胞质分裂(Cytokinesis)、细胞内物质转运、细胞周期调控及细胞凋亡等重要生理过程。Septin与肌动蛋白、微管蛋白之间有相互作用,并为SA-GEF、Rho／Rhotikine、Borges等上游分子所调控。Septin基因功能改变与一些人类疾病发生有关,包括白血病、脑肿瘤、结直肠癌、乳腺癌、肺癌等恶性肿瘤以及Parkison病、Alzheimer病等神经系统退行性疾病等。对Septin基因的深入研究将有助于加深我们对前述病理、生理过程的认识。

Two Modes of Cytoklnesis in the Generative Cells of Amaryllis

The cytokinesis process of generative cells in Amaryllis vitiata Ait. wasexamined with electron microscopy. Investigation of 14 pollen tubes showed that 70percent of the generative cells were divided by a cell plate, while the other, 30 percentshowed a cleavage furrow which occurred at the position where a cell plate should takeplace. The cell plate appeared at First as a subunit in late anaphase, which was assembledin the midzone of the phragmoplast and coalesced as one large continued unit in telophase.On the other hand, just in anaphase, as two identical chromosome masses were segregatedfrom each other, the plasma membrane of the generative cell were furrowing inside fromthe tWo sides in the interzonal region. In some instances, the cell was almost divided intotwo parts by the constriction furrow. The occurrence of cleavage seemed to be associatedwith the re-establishment of the nuclear envelope and the disorder of microtubular arrays,as well as the unusual behavior of the chromosomes.

Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determineits function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzedby indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylationbegins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phospho-H3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms “sandwich-like structure” when the chro-mosomes condensed. With the cleavage progressing, the “ladders” of the histone contract into a bigger bright dot. Thenthe histone aggregates and some of compacted microtubules in the midbody region are composed into a “bar-like”complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the “bar”,inside which locates microtubules and modified histones, to finish the cytokinesis and keep the “bar complex” out of thecells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may takepart in the formation of midbody and play a crucial role in cytokinesis.

Genotoxicity of Synthetic Food Colorants

A study was conducted to evaluate the genotoxicity, if any, of the permitted synthetic food colorants used in India. Eight synthetic food colorants namely Erythrosine （E 127）, Tartrazine （E 102）, Ponceau 4R （E 124）, Sunset Yellow FCF （E 110）, Brilliant Blue FCF （E133）, Fast Green FCF （E143）, Carmoisine （E122） and Indigo Carmine （E132） and their combination are used in sweets namely Ladu, Jilebi and Halwa in Calicut and suburban areas of Kerala, in India. The genotoxicity of the colorants alone and in combinations at different concentrations were evaluated by Cytokinesis Block Micronucleus （CBMN） Assay. It was observed that all the above colorants and their combinations could cause genotoxicity to human lymphocytes even at the permissible concentration of 100 ppm as per PFA （Prevention of Food Adulteration） Act of India. The toxicity varied from dye to dye and was proportional to their concentration. Combination of colors showed more toxicity than the individual components. Toxicity could be reduced drastically by reducing the concentration of the dyes at least 50% below the permissible limit. Permitted synthetic food colorants even at the permissible limit should be used with caution. This study demonstrated the need for redefining the permissible limit of the food colorants based on Admissible Daily Intake （ADI） as being practiced in developed countries.

The Role of CYLD in Blocking Oncogenic Cell Signaling in Melanoma

Dysregulation of components of the ubiqutin system has been linked to many diseases including melanoma. This is vital since the post-translational modification of different proteins via direct ubiquitin attachment is an important process for various cellular processes. CYLD is a tumor suppressor gene and deubiquitinating enzyme, which can remove polyubiquitin chains from their specific substrate and interfere with different signaling pathways. CYLD is frequently downregulated or even lost in melanoma cell lines or tissues compared to melanocytes. Down-regulation of CYLD leads to sustained oncogenic signaling that promotes melanoma progression and metastasis. In this review, we summarize the recent insights into the mechanisms which are responsible for the down-regulation of CYLD levels in melanoma and the signaling interactions of the CYLD gene product in melanoma. We argue that these recent insights into CYLD function invite the development of novel molecular strategies for melanoma prevention and treatment.

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean

Male sterility is an essential trait in hybrid seed production,especially for monoclinous and autogamous food crops.Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds.However,the gene responsible for the male-sterile phenotype has remained unknown.Here,we report the map-based cloning and characterization of the MS1 gene in soybean.MS1 encodes a kinesin protein and localizes to the nucleus,where it is required for the male meiotic cytokinesis after telophase Ⅱ.We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining.Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal,making them perfect tools for producing hybrid seeds.The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.

Mitotic Spindle Organization by the Preprophase Band

In higher plants, the preprophase band （PPB） of microtubules （MTs） forecasts the cell division site prior to mitosis and specifies the organization of MTs into a bipolar prophase spindle surrounding the nucleus. However, the mechanisms governing this PPB-dependent establishment of bipolarity are unclear. Here, we present evidence from live cell imaging studies that suggest a role for the MTs bridging the PPB and the prophase nucleus in mediating this function. Results from drug treatments, along with genetic evidence from null kinesin plants, suggest that these MTs contribute to the bipolarity, orientation, and position of the prophase spindle. Specifically, the absence of these bridge MTs is associated with lack of bipolarity, while non-uniform distributions of bridge MTs correlate with prophase spindle migration, deformation, and enhanced bipolarity toward the region of highest bridge MT density. This behavior does not require actomyosin-based forces, and is enhanced by suppressing MT dynamics with taxol. These observations occur during late prophase, and are coincident with the gradual closing of annular spindle poles. Based on these data, we describe a hypothetical mechanism for bridge MT-dependent organization of prophase spindles.